The last step in the production of the amyloid--3 peptide (A?), the primary component of the characteristic cerebral plaques of Alzheimer's disease (AD), is intramembranous proteolysis of the A-3 precursor protein (APR) by ?-secretase. This protease long eluded identification because of its complexity. Nevertheless, the past few years have seen considerable progress toward understanding the biochemistry of ?-secretase, and small molecule inhibitors have been indispensable tools to this end. Specifically, we and others have found that (1) ?-secretase is inhibited by substrate-based analogues containing aspartyl protease transition-state mimics;(2) the multi-pass Presenilins (PS), mutated in familial AD, contain two conserved transmembrane aspartates essential for catalysis;(3) PS is the target of transition-state analogue inhibitors of ?-secretase;(4) affinity purification of ?-secretase activity with an immobilized inhibitor isolates not only PS, but also three other components essential for activity: Nicastrin (NCT), Aph- 1, and Pen-2;(5) the protease apparently possesses an initial docking site for substrate on the outer part of an essential signaling pathway in cell differentiation and embryogenesis. ?-Secretase is a founding member of an emerging family of intramembrane proteases that apparently have their active sites embedded in the lipid bilayer. Despite the remarkable progress, much remains unknown about this unusual and important protease. Toward advancing the mechanistic understanding of ?-secretase and exploring its potential as a therapeutic target, we propose new substrates and inhibitors as molecular probes. These new molecular probes are designed to address three specific aims: (1) What is the nature of the ?- secretase active site? Of the substrate docking site? (2) How does ?-secretase recognize and handle its substrates? (3) How can ?-secretase be allosterically modulated to block proteolysis of APP but not Notch? Lay Summary: The goal of this project is to understand ?-secretase, a complex enzyme critical to the cause of Alzheimer's disease. Small organic molecules will be used as probes to characterize this enzyme, elucidate how it works, and explore its potential as a therapeutic target.